Sensitive Whole Mount Insitu Localization Of Small Rnas In Plants

Small RNAs, such as microRNAs (miRNAs), regulate gene expression and play important roles in many plant processes. Although our knowledge of their biogenesis and mode of action has significantly progressed, we still have comparatively little information about their biological functions. In particular, knowledge about their spatio-temporal expression patterns rely on either indirect detection by use of reporter constructs or labor-intensive direct detection by insitu hybridization on sectioned material. None of the current approaches allows a systematic investigation of small RNA expression patterns. Here, we present a sensitive method for insitu detection of miRNAs and siRNAs in intact plant tissues that utilizes both double-labeled probes and a specific cross-linker. We determined the expression patterns of several small RNAs in diverse plant tissues.Significance Statement Small RNAs regulate gene expression. Although their biogenesis and mode of action are largely understood, we know less about their biological functions. In particular, determining their spatiotemporal expression patterns thus far has relied on indirect detection via reporter constructs or labor-intensive insitu hybridization on sectioned material. Here, we present a quick insitu hybridization protocol for non-sectioned plant tissues, allowing semi-quantitative detection of small RNAs.